The demonstration of unequivocal and consistent binding of soluble peptidoglycan to the surfaces of macrophages is essential for the fulfillment of the major aims of the project. The disparity in the results of binding experiments may be due to unknown differences in the peptidoglycan preparations, to biological variability of the target cells or to the use of suboptimal conditions for conducting binding studies. We have improved our methods for purifying peptidoglycan polymers by the use of DEAE-Sephacel chromatography. This removes more than 95% of the protein that contaminates some of the preparations and the resulting preparations have full biological activity. To obtain homogeneous, reactive target cells, we produced several clones of the murine macrophage cell line PU5-1.8 by limiting dilution and obtained a clone, PU5-F7, that consistently responds to induction with low doses of protein-free peptidoglycan by producing colony-stimulating factors (CSF) and interleukin-1 (IL-1). In order to devise optimal conditions for binding experiments, we are performing detailed studies of the kinetics and dose responses of PU5-F7 and normal guinea pig peritoneal macrophages to protein-free peptidoglycan. By employing a serum-free, protein-supplemented medium rather than medium containing fetal calf serum, we have reduced much of the variability previously observed. Peptidoglycan in doses of 1 microgram/ml consistently induces significant levels of CSF and IL-1 by both of these target cells and maximum induction occurs with 3 microgram/ml. These doses are less than an order of magnitude than those previously estimated and indicate that binding and competitive experiments can be conducted with much less reagent than previously thought. Preliminary kinetic studies showed that significant levels of CSF were released following 9 to 12 hours of culture in serum-free conditions. In order to determine the minimum duration for induction, we are exposing cells to peptidoglycan for various times and evaluating subsequent secretion of CSF and IL-1 by cells cultured in the absence of inducers. Our initial studies show that induction times of as little as 30 minutes are sufficient to induce CSF secretion. These experiments indicate that induction occurs more rapidly than previously thought and will aid in the design of future binding studies.